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Production of 1,3-propanediol from glycerol and corn stover hydrolysate by recombinant Klebsiella pneumoniae AJ4 and Escherichia coli

초록/요약

A change from fossil-based to renewable biomass-based resources for the bio-based economy requires the development and adoption of new sustainable technologies that are more suited for the transformation of biomass feedstock to chemicals and energy. This thesis presents investigations into the development of processes based on industrial biotechnology as a key element for the production of chemicals from agriculture/industrial by-products. The chemicals of interest are the ones that could potentially serve as building blocks or platforms for other chemicals and polymers. Glycerol, a byproduct of biodiesel production, and corn stover, the non-grain part of harvested corn, were used as raw material for the production of 1,3-propanediol (1,3-PDO). In order to efficiently produce fuels from renewable resources, some microbes were isolated, medium constitutes were optimized and the wabG gene, which is involved in the lipopolysaccharides (LPS) biosynthesis, was disrupted. The microorganisms were firstly screened by using a high throughput screening method and Klebsiella pneumoniae AJ4 was isolated from soil samples, which is able to produce 1,3-PDO under aerobic conditions. To obtain the maximum 1,3-PDO titer from glycerol, the constitutes of culture medium were systematically optimized using statistical analyses based on one-factor-at-a-time (OFAT) and central composite design (CCD). Using these statistical approaches, culture medium factors were optimized, and a maximum titer of 52.59 g/L 1,3-PDO was obtained during a 26-h batch fermentation. The conversion of agricultural waste, such as corn stover, to 1,3-PDO was also considered. Corn stover was pretreated with NaOH, and then the pretreated corn stover was hydrolyzed to fermentable sugars using cellulase and cellobiase. To achieve the maximum titer of 1,3-PDO using corn stover hydrolysate (CSH), the significant factors of the culture medium were screened using Plackett-Burman design (PBD) and then optimized using CCD. Under optimal conditions, a maximum 1,3-PDO titer of 10.34 g/L was obtained during 13-h batch fermentation. The pathogenic potential from K. pneumoniae AJ4 was successfully eliminated by disrupting wabG gene involved in the LPS biosynthesis through homologous recombination. To obtain the maximum 1,3-PDO titer, the effect of initial glycerol concentration on the cell growth and effect of initial calcium carbonate (CaCO3) concentrations for pH control were investigated. Based on studies, the 1,3-PDO titer increased to 58.48 g/L with a productivity of 0.91 g/L h by K. pneumoniae AJ4-ES01 which are 78.8% and 12.3% improvement as compared to batch fermentation, respectively. Lastly the efficient production of 1,3-PDO from glycerol by engineered E. coli JM109 was achieved via the co-expression of gdrAB and addition of succinate. Construction of a dual-vector system and succinate addition during flask culture enhanced the glycerol consumption and titer of 1,3-PDO. The glycerol consumption and titer of 1,3-PDO were 86.6% and 145.6% higher, respectively, than those from the control (without co-expression of gdrAB and no addition of succinate). Under fed-batch fermentation conditions, the titer of 1,3-PDO and its conversion yield from glycerol and succinate were 13.11 g/L and 0.44 g/g, respectively.

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CONTENTS

ABSTRACT ........................................................................................................... i

CONTENTS ......................................................................................................... iv

LIST OF TABLES .............................................................................................. x

LIST OF FIGURES ......................................................................................... xii

Chapter I. General Introduction ................................................................... 1

1.1 Renewable biomass ....................................................................................... 2
1.1.1 Glycerol ............................................................................................ 4
1.1.2 Corn stover ....................................................................................... 6
1.2 1,3-Propanediol (1,3-PDO) ........................................................................... 9
1.3 Chemical synthetic methods for 1,3-PDO production ................................ 11
1.4 Biological synthetic methods for 1,3-PDO production ............................... 13
1.5 Objectives of this study ............................................................................... 15

Chapter II. Isolation of microorganisms able to produce 1,3-propanediol and optimization of medium constituents for Klebsiella pneumoniae AJ4 ..................................................................................... 17

2.1 Introduction ................................................................................................. 18
2.2 Materials and Methods ................................................................................ 20
2.2.1 HTS and microorganism isolation .................................................. 20
2.2.2 Strain identification using 16S rRNA sequencing .......................... 21
2.2.3 Effect of basal medium constituents ............................................... 22
2.2.4 Statistical method for response surface analysis ............................ 22
2.2.5 Bioreactor batch fermentation condition ........................................ 24
2.2.6 Analytical methods ......................................................................... 24
2.3 Results and Discussion ................................................................................ 26
2.3.1 Isolation using high throughput screening (HTS) and identification of strains ......................................................................................... 26
2.3.2 Effects of culture medium factors on 1,3-PDO production ............ 30
2.3.3 Optimization of the culture medium using response surface methodology (RSM) ....................................................................... 33
2.3.4 Flask culture and bioreactor batch fermentation using K. pneumoniae AJ4 ............................................................................. 39
2.4 Summary ..................................................................................................... 42
Chapter III. Optimization of culture conditions for enhanced production of 1,3-propanediol from corn stover hydrolysate by Klebsiella pneumoniae AJ4 ............................................................................ 43

3.1 Introduction ................................................................................................. 44
3.2 Material and Methods ................................................................................. 46
3.2.1 Raw materials ................................................................................. 46
3.2.2 Microorganisms and enzymes ........................................................ 46
3.2.3 Pretreatment of corn stover and enzyme hydrolysis ....................... 47
3.2.4 Effect of basal medium constituents ............................................... 50
3.2.5 Statistical method for response surface analysis ............................ 51
3.2.6 Flask culture and batch fermentation .............................................. 52
3.3 Results and Discussion ................................................................................ 54
3.3.1 Screening the effect of variable factors by PBD ............................ 54
3.3.2 Central composite design (CCD) and response surface analysis ... 57
3.3.3 Validation of the optimized condition using CSH and bioreactor fermentation .................................................................................... 65
3.4 Summary ..................................................................................................... 70

Chapter IV. Production of 1,3-propanediol from glycerol by engineered K. pneumoniae AJ4-ES01 without pathogenic potentials by inactivating lipopolysaccharide biosynthesis .................................... 71

4.1 Introduction ................................................................................................. 72
4.2 Material and Methods ................................................................................. 75
4.2.1 Bacterial strains and culture medium ............................................. 75
4.2.2 Construction of an apramycin gene cassette with long homologous region for disrupting K. pneumoniae wabG ................................... 78
4.2.3 Construction of K. pneumoniae mutant strain by using Red recombination ................................................................................. 79
4.2.4 Batch and fed-batch fermentation for 1,3-PDO production ........... 80
4.2.5 FE-SEM analysis ............................................................................ 81
4.3 Results and Discussion ................................................................................ 82
4.3.1 Construction and characterization of wabG knockout mutant ....... 82
4.3.2 1,3-PDO production by K. pneumoniae AJ4-ES01 ........................ 86
4.3.3 The effect of concentration of CaCO3 added as a neutralizing agent ............................................................................................... 90
4.3.4 Initial glycerol concentration effect on the cell growth of K. pneumoniae ..................................................................................... 92
4.3.5 Fed-batch fermentation at low glycerol concentration and pH in a 1.5-L fermentor ............................................................................... 94
4.4 Summary ..................................................................................................... 98

Chapter V. Production of 1,3-propanediol from glycerol using engineered Escherichia coli ........................................................................... 99

5.1 Introduction ............................................................................................... 100
5.2 Material and Methods ............................................................................... 103
5.2.1 Microorganisms and plasmids ...................................................... 103
5.2.2 Plasmid construction to 1,3-PDO production ............................... 103
5.2.3 Flask culture and fed-batch fermentation .................................... 107
5.3 Results and Discussion .............................................................................. 109
5.3.1 1,3-PDO productions from glycerol varied with and without p15-AB or strain specificity ....................................................................... 109
5.3.2 1,3-PDO production from CSH by JM-30BY15AB ..................... 113
5.3.3 The addition of tricarboxylic acid (TCA) cycle intermediates improved 1,3-PDO production from glycerol .............................. 117
5.3.4 1,3-PDO production by the engineered E. coli in a bioreactor ..... 121
5.4 Summary ................................................................................................... 123

Chapter VI. Overall conclusions and further research ..................... 124

Chapter VII. References ............................................................................... 128

Abstract in Korean ......................................................................................... 144

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